Published online before print December 16, 2004

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(Journal of Leukocyte Biology. 2005;77:432-437.)
© 2005 by Society for Leukocyte Biology

Detection of apoptosis by caspase-3 activation in tracheal aspirate neutrophils from premature infants: relationship with NF-B activation

Fook-Choe Cheah^*,,1, Mark B. Hampton^*, Brian A. Darlow, Christine C. Winterbourn^* and Margret C. M. Vissers^*,2

^* Departments of Pathology, Free Radical Research Group, and
Paediatrics, Christchurch School of Medicine and Health Sciences, University of Otago, New Zealand

² Correspondence: FRRG, Department of Pathology, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand. E-mail: margret.vissers{at}chmeds.ac.nz

In premature infants, inflammatory conditions in the lungs may result in the development of chronic lung disease. As neutrophil apoptosis is important for the resolution of inflammation and prevention of tissue injury, we set out to determine the extent of neutrophil apoptosis in tracheal aspirate samples from premature infants. Activation of the transcription factor nuclear factor (NF)-B, which causes a delay in neutrophil apoptosis, was also investigated. We obtained 68 tracheal aspirate samples from 27 infants with median gestation and birthweight of 26 weeks and 860 g, respectively. Apoptosis was assessed by immunofluorescent detection of the active form of caspase-3, this assay being validated with peripheral blood neutrophils. Activation of NF-B was monitored by the nuclear translocation of the p65 subunit, detected by immunofluorescence. Cleaved caspase-3 was detected in 11 of the 68 samples, and a median of 40% of the neutrophils showed activated caspase-3 (range 3–92%). A majority of the samples did not show evidence of apoptosis. Caspase activation was seen in cells with multilobed nuclear morphology, suggesting that early apoptosis was detectable. There was no significant difference in respiratory outcomes between infants with or without neutrophil apoptosis. Seventeen of the 68 samples (25%) had evidence of activated NF-B, and a median of 20% (range 6–41%) of neutrophils showed activation. In all but one tracheal aspirate sample, there was a mutually exclusive relationship between activated caspase-3 and NF-B activation, which supports in vitro observations that NF-B activation delays neutrophil apoptosis.

Key Words: activated caspase-3 • neutrophil apoptosis • chronic lung disease

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